Spatio-temporal spread of neuronal death after focal photolysis of caged glutamate in neuron/astrocyte co-cultures

Glutamate-mediated excitotoxicity is now accepted as a major mechanism of ischemic neuronal damage. In the infarct core region, massive neuronal death is observed, but neurons in the surroundings of the core (ischemic penumbra) seem viable at the time of stroke. Several hours or days after a stroke, however, many neurons in the penumbra will undergo delayed neuronal death (DND). The mechanisms responsible for such DND are not fully understood. In this study, we investigated whether and how glutamate-mediated localized excitotoxic neuronal death affects surrounding neurons and astrocytes. To induce spatially-restricted excitotoxic neuronal death, a caged glutamate was focally photolyzed by a UV flash in neuron/astrocyte co-cultures. Uncaging of the glutamate resulted in acute neuronal death in the flashed area. After that, DND was observed in the surroundings of the flashed area late after the uncaging. In contrast, DND was not observed in neuron-enriched cultures, suggesting that functional changes in astrocytes, not neurons, after focal acute neuronal death were involved in the induction of DND. The present in vitro study showed that the spatially-restricted excitotoxic neuronal death resulted in DND in the surroundings of the flashed area, and suggested that the nitric oxide (NO)-induced reduction in the expression of astrocytic GLT-1 was responsible for the occurrence of the DND.     

以葡萄糖为底物一步法合成γ-氨基丁酸整合型重组钝齿棒杆菌的构建

[目的]为了构建一株直接利用廉价的葡萄糖合成γ-氨基丁酸的重组钝齿棒杆菌,将来自于植物乳杆菌γ-氨基丁酸合成途径的关键酶谷氨酸脱羧酶基因(lpgad)在产谷氨酸菌株钝齿棒杆菌中进行整合表达,实现葡萄糖到GABA的一步法生产.[方法]运用PCR技术扩增得到带有tac启动子的谷氨酸脱羧酶基因tacgad.通过重叠PCR的方法获得钝齿棒杆菌精氨酸合成途径关键酶N-乙酰谷氨酸激酶(NAGK)基因内部缺失型基因△argB.利用自杀载体pK18mobsacB构建同源整合载体pK18-△argB::tacgad,以△argB的上下游序列为同源臂,通过两次同源重组将tacgad基因整合到钝齿棒杆菌基因组,同时将NAGK基因argB灭活,利用蔗糖致死基因sacB反向筛选标记筛选得到谷氨酸脱羧酶的重组钝齿棒杆菌C.crenatum △argB::tacgad.重组钝齿棒杆菌以葡萄糖为底物进行发酵,测定GABA含量.[结果]重组菌C.crenatum △argB::tacgad成功表达谷氨酸脱羧酶,同时阻断了精氨酸合成途径对谷氨酸到GABA代谢途径的竞争,粗酶液基本检测不到NAGK活性,发酵液无精氨酸合成.通过96 h发酵,重组菌可积累约8.28 g/L的GABA.[结论]本研究通过将谷氨酸脱羧酶基因定向整合到钝齿棒杆菌精氨酸合成途径的关键酶基因argB内部,成功表达谷氨酸脱羧酶的同时阻断竞争途径精氨酸的合成.本研究为实现直接利用葡萄糖合成GABA的一步法生产奠定了基础.     

Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor‐like (GLR) homologs are intimately associated with Ca²⁺ influx through plasma membrane and participate in various physiological processes. In pathogen‐associated molecular patterns (PAMP)‐/elicitor‐mediated resistance, Ca²⁺ fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense‐related genes by OGs. In addition, wild‐type Col‐0 plants treated with the glutamate‐receptor antagonist 6,7‐dinitriquinoxaline‐2,3‐dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs‐triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen‐mediated plant defense signaling pathways in Arabidopsis thaliana.     

Ion channels of glutamate receptors: structural modeling

Ionotropic glutamate receptors belong to the superfamily of P-loop channels as well as K⁺, Na⁺, and Ca²⁺ channels. However, the structural similarity between ion channels of the glutamate receptors and K⁺ channels is a matter of discussion. The aim of this study was to analyze differences between the structures of K⁺ channels and glutamate receptor channels. For this purpose, homology models of NMDA and AMPA receptor channels (M2 and M3 segments) were built using X-ray structures of K⁺ channels as templates. The models were optimized and used to reproduce specific data on the structure of glutamate receptor channels. Particular attention was paid to the data of the binding of channel blockers and to the results of scanning mutagenesis. The modeling demonstrates that properties of glutamate receptor channel can be reproduced assuming only local structural deformations of the K⁺ channel templates. The most valuable differences were found in the selectivity-filter region, whereas helical parts of M2 and M3 segments could have similar spatial organization with homologous segments in K⁺ channels. It is concluded that the current experimental data on glutamate receptor channels does not reveal global structural differences with K⁺ channels.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Effect of Oxidized Arachidonic Acid and Hexanal on the Mouse Taste Perception of Bitterness and Umami

The oxidization of fatty acids generates many volatile compounds forming an aroma, but little is known whether mammals use gustatory sense to detect the oxidized products as a taste or only use olfactory sense to detect as an aroma. We examined in this study the effect of aqueous extracts of the compounds from autoxidized arachidonic acid (AA) ethyl ester or hexanal which is the predominant component generated from oxidized AA by the anosmic mouse licking performance to a tastant. The addition of the water extract from oxidized AA or hexanal to a quinine hydrochloride (QHCl) solution decreased the anosmic mice licking frequency at several concentrations of QHCl. Hexanal also reduced the licking frequency of anosmic mice conditioned to avoid MSG at several concentrations of monosodium glutamate (MSG). These results suggest that hexanal would affect mouse taste perception to QHCl and MSG via the gustatory sensation.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

The Protein Encoded by NCgl1221 in Corynebacterium glutamicum Functions as a Mechanosensitive Channel

The function of the NCgl1221-encoded protein of Corynebacterium glutamicum was analyzed using Bacillus subtilis as host because a method for preparing the giant provacuole required for electrophysiological studies has been established. Expression of NCgl1221 in a strain deficient in mscL and ykuT, both of which encode mechanosensitive channels, resulted in an 8.9-fold higher cell survival rate upon osmotic downshock than the control. Electrophysiological investigation showed that the giant provacuole prepared from this strain, expressing NCgl1221, exhibited significantly higher pressure-dependent conductance than the control. These findings show that the NCgl1221-encoded protein functions as a mechanosensitive channel.     

Characterization and immobilization on nickel-chelated Sepharose of a glutamate decarboxylase A from Lactobacillus brevis BH2 and its application for production of GABA

A gene encoding glutamate decarboxylase A (GadA) from Lactobacillus brevis BH2 was expressed in a His-tagged form in Escherichia coli cells, and recombinant protein exists as a homodimer consisting of identical subunits of 53?kDa. GadA was absolutely dependent on the ammonium sulfate concentration for catalytic activity and secondary structure formation. GadA was immobilized on the metal affinity resin with an immobilization yield of 95.8%. The pH optima of the immobilized enzyme were identical with those of the free enzyme. However, the optimum temperature for immobilized enzyme was 5?°C higher than that for the free enzyme. The immobilized GadA retained its relative activity of 41% after 30 reuses of reaction within 30?days and exhibited a half-life of 19 cycles within 19?days. A packed-bed bioreactor with immobilized GadA showed a maximum yield of 97.8% GABA from 50?mM l-glutamate in a flow-through system under conditions of pH 4.0 and 55?°C.